TCRs mediate the recognition of Specific Major Histocompatibility Complex (MHC)-peptide complexes (“pMHC complexes”) which are presented as epitopes on antigen presenting cells (APC), and TCRs mediate the recognition of such pMHC epitopes by T cells. As such TCRs are essential to the functioning of the cellular arm of the immune system.
Antibodies are also known which specifically bind pMHC epitopes presented by antigen presenting cells (see for example: Neethling F A. et al., Vaccine (2008) 26 (25): 3092-102). There are antigen-binding fragment (Fab) antibodies (see for example: Chames P. et al., Proc Natl Acad Sci USA (2000) 97 (14): 7969-74; Willemsen R A. et al., J Immunol (2005) 174 (12): 7853-8; Willemsen R. et al., Cytometry A (2008) 73 (11): 1093-9) or single-chain antibody fragments (scFv) (see for example: Denkberg G. et al., J Immunol (2003) 171 (5): 2197-207; Marget M. e al., Mol Immunol (2005) 42 (5): 643-9) which specifically bind pMHC epitopes.
The native TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily which is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have quite distinct anatomical locations and probably functions. The MHC class I and class II ligands are also immunoglobulin superfamily proteins but are specialised for antigen presentation, with a highly polymorphic peptide binding site which enables them to present a diverse array of short peptide fragments at the APC cell surface.
The extracellular portion of native heterodimeric αβ TCRs consist of two polypeptides (the α and β chains) each of which has a membrane-proximal constant domain, and a membrane-distal variable domain. Each of the constant and variable domains includes an intra-chain disulfide bond. The variable domains contain the highly polymorphic loops analogous to the complementarity determining regions (CDRs) of antibodies. CDR3 of αβ TCRs interact with the peptide presented by MHC, and CDRs 1 and 2 of αβ TCRs interact with the peptide and the MHC. The diversity of TCR sequences is generated via somatic rearrangement of linked variable (V), diversity (D), joining (J), and constant genes (C).
Functional a chain polypeptides are formed by rearranged V-J-C regions, whereas β chains consist of V-D-J-C regions. The extracellular constant domain has a membrane proximal region and an immunoglobulin region. There is a single a chain constant domain, known as TRAC. The β chain constant domain is composed of one of two different β constant domains, known as TRBC1 and TRBC2 (IMGT nomenclature). There are four amino acid changes between these β constant domains. These changes are all within exon 1 of TRBC1 and TRBC2: N4K5->K5N5 and F37->Y (IMGT numbering, differences TRBC1->TRBC2), the final amino acid change between the two TCR β chain constant regions being in exon 3 of TRBC1 and TRBC2: V1->E.
A number of constructs have been devised to date for the production of recombinant TCRs. These constructs fall into two broad classes, single-chain TCRs and dimeric TCRs. Single-chain TCRs (scTCRs) are artificial constructs consisting of a single amino acid strand, which like native heterodimeric TCRs bind to MHC-peptide complexes. scTCRs can consist of a combination of TCR α and β variable regions (Vα and Vβ respectively) and TCR α and β constant regions (Cα and Cβ respectively), linked by a linker sequence (L) in several possible orientations, for example, but not limited to, the following Vα-L-Vβ, Vβ-L-Vα, Vα-Ca-L-Vβ or Vβ-Cβ-L-Vα.
A number of papers describe the production of TCR heterodimers which include the native disulphide bridge which connects the respective subunits. However, although such TCRs can be recognised by TCR-specific antibodies, none have been shown to recognise its native ligand at anything other than relatively high concentrations and/or were not stable.
In WO 03/020763 a soluble TCR is described which is correctly folded so that it is capable of recognising its native ligand, is stable over a period of-time, and can be produced in reasonable quantities. This TCR comprises a TCR α chain extracellular domain dimerised to a TCR β chain extracellular domain respectively, by means of an inter-chain disulfide bond between cysteines introduced into the constant regions of the respective chains.
Specific pMHC binding partners, ie antibodies specific for pMHC epitopes, and TCRs of both the heterodimeric and single chain type, have been proposed as targeting vectors for the delivery of therapeutic agents to antigen presenting cells. For that purpose, the therapeutic agent is required to be associated with the pMHC-binding partner in some way. Therapeutic agents which have been suggested for such targeted delivery in association with pMHC-binding partners include antibodies (see for example: Mosquera L A. et al., J Immunol (2005) 174 (7): 4381-8), cytokines (see for example: Belmont H J. et al., Clin Immunol (2006) 121 (1): 29-39; Wen J. et al. Cancer Immunol Immunother (2008) 57 (12): 1781-94), and cytotoxic agents. Where the therapeutic agent is a polypeptide, the means of association with the pMHC binding partner may be by peptidic fusion, either direct fusion or fusion via a linker sequence, to the pMHC binding partner. In those cases, there are essentially only two fusion possibilities. In the case of single chain antibodies or TCRs, fusion can in principle be at the C- or N-terminus of the TCR chain; In the case of heterodimeric antibodies or TCRs, the fusion can in principle be at the C- or N-terminus of either chain. In practice however, it appears that all known examples of pMHC binding partner-therapeutic agent fusions have been with the therapeutic agent fused to the C-terminus (see for example: Mosquera L A. et al., J Immunol (2005) 174 (7): 4381-8; Belmont H J. et al., Clin Immunol (2006) 121 (1): 29-39; Wen J. et al., Cancer Immunol Immunother (2008) 57 (12): 1781-94). This is because the functionality of an antibody or TCR, whether single chain or heterodimeric, depends on correct folding and orientation of the variable regions. Fusion of the therapeutic agent to the N-terminus of the pMHC binding partner places it ahead of one of the variable regions, and there has been an assumption in the art that the therapeutic agent located at the N-terminus will interfere with binding of the antibody or TCR to the pMHC complex, thereby reducing its binding efficiency.